Western blot
Total proteins were extracted using RIPA buffer (0.5% Nonidet P-40,
0.1% sodium deoxycholate, 150mM NaCl, 50mM Tris-Cl, pH7.5 and 1x
protease inhibitor cocktail). Cell lysates were subjected to high-speed
centrifugation at 12000xg for 15min at 4°C. Protein
concentrations were measured using Coomassie (Bradford) protein assay
kit. Total soluble proteins were then separated on 10% SDS-PAGE and
transferred into polyvinylidene difluoride membrane (Millipore,
Billerica, MA). Membrane was blocked with 5% nonfat dry milk for 1h
followed by incubation with diluted the primary antibodies (β-actin,
1:2000, Signalway antibody; Dip2a, 1:500, Novus; Dip2c, 1:1000, Abcam)
for overnight at 4°C. Then the membrane was washed in TBST for three
times, 5 min each and then incubated with secondary antibody
(anti-rabbit horseradish peroxidase conjugate, 1:5,000; anti-mouse
horseradish peroxidase conjugate, 1:5,000; Transgene) for 30min,
followed by washing three times with TBST. Signals were detected using
enhanced chemiluminescence Amersham™ ECL™ (GE Healthcare, USA) reagents.
β-actin protein served as a loading control.